We previously demonstrated that mouse ES cells, genetically labeled with GFP, have an ability to differentiate into hepatocyte-like cells in vitro. In 2004, we also investigated whether mouse ES cells could differentiate into hepatocyte-like cells in vivo. Mice were treated with carbon tetrachloride (CCl_4, intraperitoneal injection of 0.5 ml/Kg, twice a week) for 5 weeks. Following the second administration of CCl_4, 10^5 undifferentiated ES cells or 10 embryoid bodies(EBs) were transplanted into the mice spleens. Prior to transplantion, the EBs were prepared using a 5-day hanging-drop culture of 500 undifferentiated ES cells in 20 μl of culture medium without LIF. Histological evaluations were performed on post-transplantation day(PD) 10, 20, and 30. GFP-immunopositive cells were found in the periportal regions of the liver lobules on PD 10, after which those cells were found spread into the lobules on PD 20. However, they disappeared by PD 30. In CCl_4-untreated mice, GFP-immunopositive cells were not found in the liver on PD 10, 20, or 30. A double immunofluorescent study revealed that GFP-immunopositive cells were also positive for albumin, cytokeratin 18, and HNF4α. Further, the intralobular distribution of GFP-positive cells was similar between ES- and EB-transplanted mice, and ES cells that had differentiated into hepatocyte-like cells expressing albumin, cytokeratin 18, and HNF4α were observed in both. Our results suggest the possible usefulness of ES-based cell therapy for liver diseases.